The object of the proposed research is to combine the methods of physical organic chemistry, multivariate analysis and molecular graphics in the search for more effective and selective antibacterial and antiprotozoal agents, specifically antifolates.
The aim of this research is twofold - primarily to synthesize antifolates that are more potent and more selective than Trimethoprim and also to address the resistance problem by designing and synthesizing lipophilic dihydrofolate reductase (DHFR) inhibitors that retain sensitivity in resistant organisms. These inhibitors will encompass two classes of compounds such as 2,4-dihydroxy-5-bromo-7-(methyl-(2,4-diamino-pyrimidinyl)-quinolines Ia and 2,4-diamino-5-(3'-bromo, 4'-carboxamidomethoxy, 5'-acylamino-benzyl) pyrimidine IIb. The proposed chemical structures have been based on a careful analysis of QSAR models derived from a study of approximately 68 2,4-diamino-5-X-benzyl pyrimidines III and their interactions with both chicken liver DHFR and E. coli DHFR. These studies have been augmented by molecular graphic analysis of these inhibitors with the above mentioned enzymes whose X-ray crystallographic coordinates are known at 2.5 angstrom resolution. These antifolates will be synthesized by utilizing well defined procedures and their purities established by HPLC if warranted. Their inhibitory activities versus chicken liver DHFR. E. coli DHFR and L. major DHFR will be assessed using an established spectrophotometric method which increases DHFR activity with and without added inhibitor. It involves the oxidation of NADPH to NADP+ and the reduction of dihydrofolic acid to tetrahydrofolic acid at 340 nm using a Durum stopped flow spectrophotometer. The inhibition constants (Ki) and their 95 percent confidence limit will then be calculated by using an """"""""in house"""""""" DHFR software program """"""""jackknife."""""""" The techniques developed with these DHFR inhibitors should yield valuable insights regarding the binding modes of flexible and rigid ligands with DHFR and their optimization of hydrophobic/hydrophilic points of contact at the molecular level. These enzyme inhibitors will also be evaluated in cellular systems (E. coli cultures sensitive and resistant to methotrexate).
